Project Summary/Abstract Laser ablation electrospray ionization mass spectrometry (LAESI-MS) is a powerful method for ambient mass spectrometry imaging that does not require vacuum or sample preparation. However, LAESI is limited in sensitivity and mass range due to the limited ability to efficiently produce nanoparticles that are required for merging with the electrospray to create ions. Instead, most of the material ablated is lost as large microparticle pieces of tissue. Improving the efficiency of laser ablation of nanoparticles is necessary to achieve high sensitivity for LAESI imaging. The goal of this proposed research is to increase the efficiency of nanoparticle ablation for LAESI mass spectrometry by more than a factor of ten using combined infrared and ultraviolet laser ablation. The elastin and collagen in skin tend to hold it together when a pulsed laser hits it. Increasing the laser energy removes more material but it comes out as large chunks. This limits the precision of the ablation and for mass spectrometry analysis, the large pieces of tissue do not result in ion formation. Two-laser ablation can overcome this problem and can both improve the precision of the ablation as well as increase the efficiency of nanoparticle formation. Two lasers, one in the ultraviolet wavelength region and the other in the infrared are focused onto the same spot of a tissue section. The UV laser is fired first to break bonds in the tissue extracellular material and to produce small gas molecules that will serve as explosive boiling nuclei when the infrared laser is fired. When the IR laser hits the tissue, the explosive phase change removes the weakened tissue as nanoparticles, which merges with the electrospray to form ions. The two-laser system will be used with a two-dimensional translation stage for mass spectrometry imaging of tissue. The potential improvement in limit of detection for tissue imaging is a factor of ten or more. The two-laser ablation approach has applications in other areas of mass spectrometry imaging. For example, matrix-assisted laser desorption ionization (MALDI) imaging requires tissue break-up for analysis in high- resolution transmission geometry (back side irradiation) mode. Inductively coupled plasma (ICP) mass spectrometry requires particle break-up for efficient atomization and ion formation. This applies as well to the mass cytometry method of antibody tagging for mass spectrometry. The approach also has the potential to improve the efficiency and precision of ablation in surgical applications.